This project seeks to determine specific changes in the genome induced by chemical carcinogens and their relevance to the initiation and development of neoplasia. (1) To explore if different transforming DNA sequences are activated by different carcinogens, DNA from BALB/3T3 cells transformed by several chemical carcinogens and by U.V. light was transfected in NIH/3T3 cells. Three positive benzo[a]pyrene(BP)-transformed lines were analyzed for sensitivity to the restriction endonucleases, EcoRI, HindIII, BamHI and XbaI, and showed different patterns of sensitivity with no evidence of rearrangement or amplification of ras oncogenes. However, further analysis with MspI revealed a polymorphism in the v-Ha-ras homologous sequences. Since a MspI polymorphism at the 12 codon of the human and the rat c-Ha-ras gene distinguishes the malignant from the normal alleles, these results suggest that the transforming gene may be a point mutated allele of c-Ha-ras. Further work is under way to investigate the mechanisms responsible for different restriction enzyme sensitivity patterns of transforming DNAs inducted by BP. (2) The molecular basis for the frequent activation of c-Ha-ras genes by point mutations is being explored. The binding of BP to hamster liver DNA reached a maximum shortly after injection of BP into the portal vein. A major portion of this BP binding occurred in DNAase I hypersensitive regions which are correlated with putative regulatory regions of actively expressed genes. These results suggest that persistent adducts may be located within the coding region and therefore may be relevant to gene activation by point mutation. (3) To establish the molecular events underlying the mechanisms of tumor promotion, two apparently new transforming genes, required for tumor promotion in the JB-6 mouse epidermal cell lines, were cloned from promoter-sensitive preneoplastic cells. Studies are under way on their structure and DNA sequence and on their presence and function in normal mouse keratinocytes. Human homologs of these genes are being isolated from a genomic library and their structure and function are being studied.